Extravascular Tissue Research Articles

Ultrasound-activated microbubbles mediate F-actin disruptions and endothelial gap formation during sonoporation

Locally opening up the endothelial barrier in a safe and controlled way is beneficial for drug delivery into the extravascular tissue. Although ultrasound-induced microbubble oscillations can affect the endothelial barrier integrity, the mechanism remains unknown. Here we uncover a new role for F-actin in microbubble-mediated endothelial gap formation. Unique simultaneous high-resolution confocal microscopy and ultra-high-speed camera imaging (10 million frames per second) reveal that single oscillating microbubbles (radius 1.3–3.8 μm; n = 48) induce sonoporation in all cells in which F-actin remodeling occurred. F-actin disruption only mainly resulted in tunnel formation (75 %), while F-actin stress fiber severing and recoil mainly resulted in cell-cell contact opening within 15 s upon treatment (54 %) and tunnel formation (15 %). F-actin stress fiber severing occurred when the fibers were within reach of the microbubble's maximum radius during oscillation, requiring normal forces of ≥230 nN. In the absence of F-actin stress fibers, oscillating microbubbles induced F-actin remodeling but no cell-cell contact opening. Together, these findings reveal a novel mechanism of microbubble-mediated transendothelial drug delivery, which associates with the underlying cytoskeletal F-actin organization.

Activation of autoreactive lymphocytes in the lung by radioresistant cells expressing a STING gain-of-function mutation.

Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in infancy (SAVI). Patients with SAVI develop interstitial lung disease (ILD) and produce autoantibodies that are commonly associated with systemic autoimmune diseases. Mice expressing the most common SAVI mutation, STING V154M (VM), similarly develop ILD but exhibit severe T and B cell lymphopenia and low serum Ig titers, and they lack autoantibodies. Importantly, lethally irradiated VM hosts reconstituted with WT stem cells (WT→VM) still develop ILD. In this study, we find that WT→VM chimeras had restored B cell function, produced autoantibodies, and thereby recapitulated the loss of tolerance seen in patients with SAVI. Lymphocytes derived from both WT and BCR or TCR transgenic (Tg) donors accumulated in the extravascular lung tissue of WT+Tg→VM mixed chimeras, but lymphocyte activation and germinal center formation required WT cells with a diverse repertoire. Furthermore, when T cells isolated from the WT→VM chimeras were adoptively transferred to naive Rag1-deficient secondary hosts, they trafficked to the lung and recruited neutrophils. Overall, these findings indicated that VM expression by radioresistant cells promoted the activation of autoreactive B cells and T cells that then differentiated into potentially pathogenic effector subsets.

Abstract B059: A novel pancreatic cancer mouse model with human immunity

Abstract The iRGD tumor-penetrating peptide delivers chemically linked drugs and even co-injected free drugs selectively to extravascular tumor tissue by targeting the αvβ5 integrin and neuropilin-1 (NRP1). A phase 1b clinical trial showed that iRGD appears to safely double the response rate of standard chemotherapy in patients with stage 4 pancreatic ductal adenocarcinoma (PDAC). We recently found that long-term iRGD treatment, even as a monotherapy, reduced regulatory T cells (Tregs) selectively in the PDAC tissue, which led to the expansion of CD8+ T cells and improved efficacy of immunotherapy in syngeneic PDAC mice. This finding led to the discovery of αvβ5 integrin being a novel targetable marker for tumor-resident Tregs in PDAC mice. Our recent data show that αvβ5 integrin+ Tregs are also present in human PDAC tissue. To study the significance of αvβ5 integrin+ human Tregs as a therapeutic target for iRGD, we developed a humanized PDAC (huPDAC) mouse model by transplanting human PDAC cells into human immune system (HIS) mice, which harbor fully matured human CD8+ and CD4+ T cells, natural killer T cells, B cells, and dendritic cells. The CD8+ T cells are human leukocyte antigen (HLA)-restricted and show a tumor antigen-specific response. We orthotopically implanted HLA-matched human PDAC cells into HLA-A2-positive HIS mice. Both an established human PDAC cell line (PANC-1) and patient-derived PDAC cells (0779E) successfully formed orthotopic tumors and liver metastases. The tumors were infiltrated by various human immune cells at a ratio similar to that found in patient-derived PDAC tissues. Of note, αvβ5 integrin+ human Tregs were also noted in the tumors but not in the spleen. iRGD monotherapy increased the ratio of CD8+ T cells per Tregs in the tumors, suggesting that iRGD will likely function as an immunomodulator in humans. The first patient was recently dosed in a new phase 1b/2a clinical trial (iLSTA Trial: ACTRN12623000223639), which assesses the safety and preliminary efficacy of iRGD in combination with standard-of-care chemotherapy and immunotherapy in locally advanced PDAC patients. Citation Format: Norio Miyamura, Kodai Suzuki, Richard A. Friedman, Aristidis Floratos, Yuki Kunisada, Kazuya Masuda, Andrew M. Lowy, Moriya Tsuji, Kazuki N. Sugahara. A novel pancreatic cancer mouse model with human immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B059.

Thrombopoietin mimetic therapy alleviates radiation-induced bone marrow vascular injury in a bone marrow transplant mouse model.

There is a need for therapies that can mitigate bone marrow dysfunction and organ toxicity that occur following myeloablative injury and reduced intensity conditioning regimens used in patients undergoing bone marrow transplantation (BMT). The pathogenesis of adverse effects from BMT conditioning has been linked to injury to the vascular endothelium, bone marrow (BM), and other organs. To evaluate the impact of the thrombopoietin mimetic drug JNJ-26366821 (TPOm) on BM vascular recovery in mice undergoing myeloablative radiation conditioning followed by BMT. TPOm (doses: 0 µg, 300 µg, 1000 µg per Kg body weight) was administered on Days 0 and 7 after BMT, in mice receiving a total body irradiation (TBI) conditioning regimen (5.5 Gy x 2) before congenic BMT. BM donner cell engraftment was analyzed using flow cytometry on Days 7, 14, and 30 post-BMT. The morphological and biophysical properties of the BM vasculature were evaluated by intravital multiphoton microscopy (MPM) and immunofluorescence confocal imaging. Herein, morphological properties involve microvascular density (MVD), vessel diameter, and vascular area, while biophysical properties include transfer rate (Ktrans) of contrast within the BM vascular niche, as well as the fractional volume (vec ) of extracellular extravascular tissue (EES). No significant difference in donor chimerism was observed at days 7, 14, and 30 post-BMT, between TPOm and PBS-treated mice. TPOm intervention improved BM vasculature regeneration in transplanted mice. The MVD, Ktrans, and BM vasculature as well as vascular endothelial growth factor receptor-2 (VEGFR2) in the BM, showed a dose dependent improvement in mice treated with TPOm. On day 14 post-BMT, the group receiving 1000 µg/Kg TPOm showed significant shifts (p-value < 0.05) in MVD, Ktrans, and VEGFR2 expression from their corresponding control types (TPOm dose 0 µg) towards levels comparable to healthy controls. TPOm intervention augments BM vascular structure and function, which may be important for hematopoietic recovery and bone marrow function in radiation conditioned hematopoietic stem cell transplant patients, in addition to enhancing platelet recovery.

Hemophilic Pseudotumor of Paranasal Sinuses in a Child with Severe Hemophilia a Receiving Emicizumab: A Case Report

Emicizumab, a bispecific humanized monoclonal antibody, bridges activated factor IX (FIX) and FX to restore the function of missing activated FVIII in individuals with hemophilia A (PwHA). Prophylaxis with subcutaneous emicizumab promotes adequate hemostasis in PwHA. Emicizumab prophylaxis in children with hemophilia A has been approved in the United States for use in PwHA ages newborn and older, with or without FVIII inhibitors. We report a 2-year-old boy with severe congenital hemophilia A who initially received prophylaxis with standard half-life factor VIII but later switched to subcutaneous emicizumab at 6 mg/kg every four weeks due to difficult IV access and parental preference. Shortly after starting emicizumab, the patient presented to the hospital with severe episodic epistaxis from the left nostril, requiring ICU admission and ENT consultation. Despite treatment, the condition did not improve, prompting the need for imaging studies. Subsequently, the patient developed ipsilateral eye proptosis and a mass protruding from the left nostril. CT and MRI imaging of the orbit revealed an expansible soft tissue mass measuring 2 x 2.5 x 4 cm occupying the right sphenoid sinus, extending to the nasal cavity. The mass had caused erosion of the clivus and the anterior cranial fossa floor, extending to the middle cranial fossa (Figures 1 &amp; 2). He underwent endoscopic nasal debulking of the left nostril mass, and the pathological examination revealed a cystic mass with a thick capsule and intra-cystic blood clots, consistent with the characteristics of a hemophilic pseudotumor (HP). The fibrous stroma contained hemosiderin-laden macrophages with no evidence of malignancy. Emicizumab was discontinued before the operation, and the patient received several doses of recombinant FVIII to maintain hemostasis. Despite the debulking procedure, the patient continued to experience episodic epistaxis and joint bleeding. An inhibitor assay was performed at that time and showed high levels, leading to a switch to on-demand FVIII treatment for hemophilia A with an inhibitor. Managing the bleeding episodes with bypassing agents proved challenging, and the family expressed dissatisfaction. A decision was made to resume emicizumab treatment, and the patient eventually stabilized, experiencing no bleeding for nearly six months. Additionally, he regularly follows up with ENT specialty, with no further intervention or progression of the tumor. Notably, he did not receive any bypassing agents concomitantly with emicizumab. HP is characterized by encapsulated blood clots accumulating in extravascular tissues, especially the musculoskeletal system, in patients with bleeding tendencies, particularly hemophiliacs. It is a benign, expansible, soft tissue-like mass that exerts pressure on surrounding structures, mimicking a locally invasive malignancy. HP is a hematoma that grows and causes bone erosion through repeated bleeding episodes. A literature review revealed that HP is a rare hemorrhagic complication that develops in 1-2% of patients with severe hemophilia (Magallón et al. Am J Hematol. 1994). Its occurrence in patients receiving emicizumab prophylaxis is extremely rare. The first reported case involved a Japanese child with hemophilia who presented with a pseudotumor occupying the left maxillary sinus after repeated episodes of epistaxis. The mass caused a downward deviation of the ipsilateral eye and bulged into the buccal cavity. The patient received monthly prophylaxis with emicizumab at 6 mg/kg (Kawahara et al. Int J Hematol. 2022), but unlike our patient, he received bypassing agents concomitantly. Our reported case is unique because HP developed independently of bypassing agent therapies, as the patient did not receive any bypassing agents before the tumor's development. The likely explanation is related to repeated bleeding insults to the same anatomical location, leading to the cumulative development of hematoma and HPs. Another possibility could be attributed to the known adverse thrombotic effects of emicizumab treatment. Physicians treating hemophilia should remain vigilant for abnormal clinical signs, such as new masses in patients receiving emicizumab, which could indicate HP.

Targeted delivery of hybrid nanovesicles for enhanced brain penetration to achieve synergistic therapy of glioma

Blood-brain barrier (BBB) obstructing brain drug delivery severely hampers the therapeutic efficacy towards glioma. An efficient brain delivery strategy is of paramount importance for the treatment of glioma. Inspired by brain targeting exosome, biomimetic BBB penetrated hybrid (pHybrid) nanovesicles, engineered by membrane fusion between blood exosome and tLyp-1 peptide modified liposome, is explored for brain targeting drug delivery. Transferrin receptor (TfR) on pHybrid nanovesicles facilitates the BBB transcytosis into brain parenchyma, and eventually endocytosed by glioma cells and diffusion to extra-vascular tumor tissues under the guidance of tLyp-1 peptide. pHybrid nanovesicles co-loaded with salvianolic acid B (SAB) and cryptotanshinone (CPT), which is constructed by membrane hybridization of blood exosome loaded with SAB and tLyp-1 modified liposome loaded with CPT, are explored for cytotoxic and anti-angiogenetic therapy towards glioma. Upon accumulation at tumor site, the loaded CPT and SAB shows synergistic effects towards glioma from cytotoxicity on cancer cells and anti-angiogenesis on tumor, respectively. Overall, this study provides a biomimetic nanoplatform for increased BBB transcytosis into brain parenchyma, which serves as a prospective strategy for delivering therapeutic agents against glioma through synergistic mechanisms.

Pharmacodynamic Age Structured Population Model For Cell Trafficking

ObjectivesCell trafficking encompasses movement of the immune system cells (e.g., granulocytes, lymphocytes) between the blood and the extravascular tissues (e.g., lymph nodes). Corticosteroids are known to suppress cell trafficking. The age-structured cell population models introduce the transit time as a structure that allows one to quantify the distribution of times the immune cells spend in the blood and the extravascular tissues. The objective of this work is to develop an age-structured cell population model describing drug effects on cell trafficking and to implement the model in pharmacometric software to enable parameter estimation and simulations. MethodsWe adopted the well-known McKendrick age-structured population model to describe the age distributions in two cell populations: blood cells and cells in the extravascular space. The hazard of cell recirculation from the extravascular tissues was age dependent and described by the Weibull function with the shape ν and scale β parameters. The drug effect on cell trafficking was modeled as the product of the Emax function of the drug plasma concentration and the Weibull hazard. The model was implemented in NONMEM 7.5.1. The model was applied to the basophil data in 34 healthy subjects who received a single intramuscular or oral dose of 6 mg dexamethasone (DEX). A recently published pharmacokinetic model was applied to describe DEX plasma concentration. Typical values of parameter estimates were further used to simulate the DEX effect of the basophil mean transit time in the extravascular tissues. ResultsSimulations of basophil time courses for varying ν demonstrated that the rebound in the blood count data following drug administration is only possible for ν >1. The estimates of model parameters were ν = 3.02, β = 0.00863 1/h, and IC50 = 7.47 ng/mL. The calculated baseline mean transit times of basophils in the blood 7.2 h and extravascular tissues 104.9 h agree with the values reported in the literature. ConclusionsWe introduced an age-structured population model to describe cell trafficking between the blood and extravascular tissues. The model was adopted to account for the inhibitory drug effect on the cell recirculation. We showed that the age structure is essential to explain the rebound observed in the blood count response to a single dose drug administration. The model was validated using the basophil responses to DEX treatment in healthy subjects.

Serum opacity factor normalizes erythrocyte morphology in Scarb1−/− mice in an HDL-free cholesterol-dependent way

Compared with WT mice, HDL receptor-deficient (Scarb1−/−) mice have higher plasma levels of free cholesterol (FC)-rich HDL and exhibit multiple pathologies associated with a high mol% FC in ovaries, platelets, and erythrocytes, which are reversed by lowering HDL. Bacterial serum opacity factor (SOF) catalyzes the opacification of plasma by targeting and quantitatively converting HDL to neo HDL (HDL remnant), a cholesterol ester-rich microemulsion, and lipid-free APOA1. SOF delivery with an adeno-associated virus (AAVSOF) constitutively lowers plasma HDL-FC and reverses female infertility in Scarb1−/− mice in an HDL-dependent way. We tested whether AAVSOF delivery to Scarb1−/− mice will normalize erythrocyte morphology in an HDL-FC-dependent way. We determined erythrocyte morphology and FC content (mol%) in three groups—WT, untreated Scarb1−/− (control), and Scarb1−/− mice receiving AAVSOF—and correlated these with their respective HDL-mol% FC. Plasma-, HDL-, and tissue-lipid compositions were also determined. Plasma- and HDL-mol% FC positively correlated across all groups. Among Scarb1−/− mice, AAVSOF treatment normalized reticulocyte number, erythrocyte morphology, and erythrocyte-mol% FC. Erythrocyte-mol% FC positively correlated with HDL-mol% FC and with both the number of reticulocytes and abnormal erythrocytes. AAVSOF treatment also reduced FC of extravascular tissues to a lesser extent. HDL-FC spontaneously transfers from plasma HDL to cell membranes. AAVSOF treatment lowers erythrocyte-FC and normalizes erythrocyte morphology and lipid composition by reducing HDL-mol% FC.

Effect of Astragali radix extract on pharmacokinetic behavior of dapagliflozin in healthy and type 2 diabetic rats.

Objective: This study aimed to investigate effect of antidiabetic herb Astragali Radix (AR) on pharmacokinetic behavior of dapagliflozin (DAPA) in healthy rats and type 2 diabetes mellitus (T2DM) rats. Methods: The T2DM rats were induced by high-fat diet (HFD) and intraperitoneal injection of streptozotocin (STZ). Concentrations of DAPA in healthy and T2DM rat plasma were determined by UPLC-MS/MS method. Effect of AR extract (ARE) on pharmacokinetic behavior of DAPA in healthy and T2DM rats was evaluated, respectively. Results: The diabetes status and co-administrated with ARE significantly affected pharmacokinetic behaviors of DAPA in the rats. Compared to that in healthy rats, t max of DAPA significantly shortened, its C max significantly increased in T2DM rats, and its t 1/2, V, AUC, CL and MRT kept unchanged. When ARE was co-administrated with DAPA, C max of DAPA significantly increased, its t max and MRT significantly decreased, and its t 1/2, V, AUC and CL kept unchanged in healthy rats. t max and C max of DAPA significantly decreased, its t 1/2 and V significantly increased, and its AUC, CL and MRT were unchanged in T2DM rats when ARE was co-administrated with DAPA. Co-administration of DAPA and ARE promoted absorptive rate of DAPA, increased its extravascular tissue distribution, and prolonged its duration of action. ARE did not cause accumulation of DAPA in vivo. Conclusion: Both disease status of T2DM and co-administration of ARE affect pharmacokinetic behavior of DAPA in vivo. Potential pharmacokinetic interactions may occur in vivo when herbs and drugs are co-administrated, which may affect efficacy and safety of drugs.

Gallation and B-Ring Dihydroxylation Increase Green Tea Catechin Residence Time in Plasma by Differentially Affecting Tissue-Specific Trafficking: Compartmental Model of Catechin Kinetics in Healthy Adults.

Catechins in green tea extract (GTE) (epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin (EC), epicatechin gallate (ECG)) vary in bioactivity. We developed a physiologically relevant mathematical model of catechin metabolism to test the hypothesis that fractional catabolic rates of catechins would be differentially affected by their structural attributes. Pharmacokinetic data of plasma and urine catechin concentrations were used from healthy adults (n = 19) who ingested confections containing 0.5 g GTE (290 mg EGCG, 87 mg EGC, 39 mg EC, 28 mg ECG). A 7-compartmental model of catechin metabolism comprised of the gastrointestinal tract (stomach, small and large intestine), liver, plasma, extravascular tissues, and kidneys was developed using a mean fraction dose of EGCG, ECG, EGC, and EC. Fitting was by iterative least squares regression analysis, and goodness of fit was ascertained by the estimated variability of parameters (FSD < 0.5). The interaction of gallation and B-ring dihydroxylation most greatly extended plasma residence time such that EGC > EC = EGCG > EGC. The interaction between gallation and B-ring dihydroxylation accelerated the transfer from the upper gastrointestinal tract to the small intestine but delayed subsequent transfers from the small intestine through the liver to plasma and from kidneys to urine. Gallation and B-ring dihydroxylation independently delayed the transfer from plasma to extravascular tissues, except the uptake to kidneys, which was slowed by gallation only. This multi-compartment model, to be validated in a future study, suggests that gallation and B-ring dihydroxylation affect catechin catabolism in a tissue-specific manner and thus their potential bioactivity.

Preclinical Pharmacokinetics and CYP Modulation Activity of Chebulinic Acid: A Potent Molecule Against Metabolic Disease.

Chebulinic acid (CA) is an active constituent of Terminalia chebula fruits with therapeutic potential against multiple metabolic diseases, including dementia, benign prostate hyperplasia, and osteoporosis. The present work intends to explore the preclinical pharmacokinetics, including the absolute bioavailability of CA and its influence on the gene expression of cytochrome P450 enzymes in the liver. Quantifying CA and probe drugs in vitro samples and preclinical serum samples of male SD rats were performed using LC-MS/MS. The influence of CA on the hepatic CYPs and their gene expression was analyzed in rat liver by quantitative real-time polymerase chain reaction. The plasma protein binding was found to be 84.81 ± 7.70 and 96.34 ± 3.12, blood-to-plasma ratio of 0.62 ± 0.16 and 0.80 ± 0.23 at 1 μM and 10 μM concentrations, respectively. Again, the absolute oral bioavailability of CA at 100 mg/kg was found to be 37.56 ± 7.3%. The in-vivo pharmacokinetic profile of probe drugs revealed CA to have significant inducing effects on CYP1A2, 2C11, 2D2, and 2E1 after 14 days, which correlates to both in-vitro rat microsomal data and gene expression results. Altogether, pharmacokinetic parameters reveal CA to have an affinity to distribute across different extravascular tissues and induce rat liver CYP enzymes.

Mesh-free high-resolution simulation of cerebrocortical oxygen supply with fast Fourier preconditioning.

Oxygen transfer from blood vessels to cortical brain tissue is representative of a class of problems with mixed-domain character. Large-scale efficient computation of tissue oxygen concentration is dependent on the manner in which the tubular network of blood vessels is coupled to the tissue mesh. Models which explicitly resolve the interface between the tissue and vasculature with a contiguous mesh are prohibitively expensive for very dense cerebral microvasculature. We propose a mixed-domain mesh-free technique whereby a vascular anatomical network (VAN) represented as a thin directed graph serves for convection of blood oxygen, and the surrounding extravascular tissue is represented as a Cartesian grid of 3D voxels throughout which oxygen is transported by diffusion. We split the network and tissue meshes by the Schur complement method of domain decomposition to obtain a reduced set of system equations for the tissue oxygen concentration at steady state. The use of a Cartesian grid allows the corresponding matrix equation to be solved approximately with a fast Fourier transform-based Poisson solver, which serves as an effective preconditioner for Krylov subspace iteration. The performance of this method enables the steady-state simulation of cortical oxygen perfusion for anatomically accurate vascular networks down to single micron resolution without the need for supercomputers.

Abstract 406: Normalization Of Erythrocyte Morphology In Scarb1 -/- Mice By Bacterial Serum Opacity Factor Is Dependent On HDL-free Cholesterol

Compared to wild-type mice, HDL-receptor-deficient (Scarb1 -/- ) mice have higher plasma levels of free cholesterol (FC)-rich HDL and exhibit multiple pathologies—female infertility and deranged platelet and erythrocyte morphology and function. These pathologies are associated with a low mol% FC in ovaries, platelets, and erythrocytes, effects that are reversed by the HDL-lowering drug probucol. Some strains of Streptococcus pyogenes secrete a protein, serum opacity factor (SOF), which catalyzes the clouding i.e., opacification, of plasma. SOF specifically targets and quantitatively converts HDL into three products, that include a cholesteryl ester-rich microemulsion containing all the cholesterol content of &gt;100,000 HDL particles as well as APOE and its heterodimer with APOA2 as its sole apolipoproteins. Delivery of SOF with an adeno-associated virus (AAV SOF ) constitutively lowers plasma HDL-FC and reverses female infertility in Scarb1 -/- mice. Thus, we tested the hypothesis—AAV SOF delivery to Scarb1 -/- mice will normalize erythrocyte morphology in an HDL-FC dependent way. The erythrocyte morphology and FC content expressed as mol% FC of three groups of mice—WT, untreated Scarb1 -/- mice (control) and Scarb1 -/- mice receiving AAV SOF —were compared and correlated with their respective HDL-mol% FC. Among Scarb1 -/- mice, AAV SOF treatment normalized reticulocyte number, erythrocyte morphology and erythrocyte mol% FC. Plasma- and HDL-mol% FC positively correlated (p &lt; 0.0001) across all three groups of mice. HDL-mol% FC also positively correlated with erythrocyte mol% FC (p &lt; 0.0001). Finally, the erythrocyte mol% FC positively correlated with both the number of reticulocytes (p = 0.006) and abnormal erythrocytes (p &lt; 0.0001). Although less profound, AAV SOF treatment also reduced the FC contents of some extravascular tissues. HDL-FC spontaneously transfers from plasma lipoproteins to cell membranes in multiple tissues sites on a time scale of minutes to a few hours. FC-enrichment of erythrocytes, which are in contact with plasma HDL, was more profound than FC enrichment in tissues. AAV SOF treatment lowers both plasma HDL-FC and erythrocyte-FC and normalizes erythrocyte morphology and lipid composition in an HDL-FC dependent way.

Vascular space occupancy asymmetric spin echo (VASO-ASE) for non-invasive quantification of cerebral oxygen extraction fraction.

Asymmetric spin echo (ASE) MRI is a method for measuring regional oxygen extraction fraction (OEF); however, extravascular tissue models have been shown to under-estimate OEF. The hypothesis investigated here is that the addition of a vascular-space-occupancy (VASO) pre-pulse will more fully suppress blood water signal and provide global OEF values more consistent with physiological expectation and 15 O positron emission tomography (PET)-validated T2 -relaxation-under-spin-tagging (TRUST) OEF measures. Healthy adults (n= 14; age=27.7 ± 5.2y; sex=7/7 male/female) were scanned at 3.0T. Multi-echo ASE without inter-readout refocusing (ASERF- ), multi-echo ASE with inter-readout refocusing (ASERF+ ), and single-echo VASO-ASE were acquired twice each with common spatial resolution=3.44 × 3.44 × 3.0 mm and τ= 0-20 ms (interval=0.5ms). TRUST was acquired twice sequentially for independent global OEF assessment (τCPMG = 10ms; effective TEs=0, 40, 80, and 160 ms; spatial resolution=3.4 × 3.4 × 5 mm). OEF intraclass-correlation-coefficients (ICC), summary statistics, and group-wise differences were assessed (Wilcoxon rank-sum; significance: two-sided p< 0.05). ASERF+ (OEF=36.8 ± 1.9%) and VASO-ASE (OEF=34.4 ± 2.3%) produced OEF values similar to TRUST (OEF=36.5 ± 4.6%, human calibration model; OEF=32.7 ± 4.9%, bovine calibration model); however, ASERF- yielded lower OEF (OEF=26.1 ± 1.0%; p< 0.01) relative to TRUST. VASO-ASE (ICC=0.61) yielded lower ICC compared to other ASE variants (ICC >0.89). VASO-ASE and TRUST provide similar OEF values; however, VASO-ASE spatial coverage and repeatability improvements are required.

Modification of PEG reduces the immunogenicity of biosynthetic gas vesicles.

Nanobubbles have received great attention in ultrasound molecular imaging due to their capability to pass through the vasculature and reach extravascular tissues. Recently, gas vesicles (GVs) from archaea have been reported as acoustic contrast agents, showing great potential for ultrasound molecular imaging. However, the immunogenicity and biosafety of GVs has not yet been investigated. In this study, we examined the immune responses and biosafety of biosynthetic GVs and polyethylene glycol (PEG)-modified GVs (PEG-GVs) in vivo and in vitro. Our findings suggest that the plain GVs showed significantly stronger immunogenic response than PEG-GVs. Less macrophage clearance rate of the RES and longer circulation time were also found for PEG-GVs, thereby producing the better contrast imaging effect in vivo. Thus, our study demonstrated the PEG modification of biosynthetic GVs from Halobacterium NRC-1 is helpful for the future application of GVs in molecular imaging and treatment.

Non-contrast estimate of blood-brain barrier permeability in humans using arterial spin labeling and magnetization transfer at 7T.

Blood-brain barrier (BBB) dysfunction is associated with a number of central nervous system diseases. This study demonstrates the application of a novel noninvasive technique to measure the BBB permeability in the human brain at 7T. The technique exploits the fact that, when tissue macromolecules are saturated by off-resonance RF pulse, the intravascular and the extravascular (tissue) water experience different magnetization transfer effects. This principle was combined with arterial spin labeling to distinguish between the intravascular and the tissue water, and was used to calculate perfusion, water extraction fraction (E), and BBB permeability surface area product for water (PS). Simultaneous coregistered magnetization transfer ratio maps were also generated that can provide valuable additional information. Eighteen healthy volunteers (seven females), age = 27 ± 11 years and weight = 65 ± 9kg, participated in the study. Average perfusion was 67 ± 5 and 29 ± 4ml/100 g/min (p< 0.05); and E was 0.921 ± 0.025 and 0.962 ± 0.015 (p < 0.05) in the gray matter (GM) and the white matter (WM), respectively. PS was higher in the GM (171 ± 20 ml/100 g/min) compared with the WM (95 ± 18 ml/100 g/min) (p < 0.05). The parameters exhibited good reliability with test re-test experiments. The sensitivity of this technique was demonstrated by 200 mg caffeine intake, which resulted in a decrease in the resting PS by ~31%.

Expression of selected cytokeratins in human placenta - a preliminary observational study

Abstract Every human body is made up of billions of cells, and every cell consists of thousands of microscopic structures. Thanks to the presence of the cytoskeleton, which is built by microfilaments, microtubules, and intermediate filaments, cells are able to fulfill their main function. Dozens of genes encode a large family of cytoskeletal proteins, which form 10-nanometer-long microfilaments, called cytokeratins. The study was carried out on seven mature human placentas without significant pathology - all eligible mothers were healthy. The collection of basic anthropometric data preceded the dissection of the placentas. Paraffin blocks were made in the usual manner, and hematoxylin and eosin-stained slides were made afterward. Immunohistochemical reactions were performed and the expression of the studied markers was evaluated independently by two observers. Evaluation of microscopic material revealed the absence of expression of antibodies for cytokeratin 5/6 in placental tissues. Moreover, strong expression of cytokeratin 7 was demonstrated in the villi trophoblast in all types of villi. Immunohistochemical reactions were observed in the mesenchyme within the blood vessel wall, as well as in the extravascular tissue. The human placenta is an organ that only exists during the intrauterine period of human development and undergoes rapid changes and dynamic growth during pregnancy. These types of processes define selected placental cells as ‘pseudo-tumorigenic tissue’ because of the numerous similarities trophoblast cells have to tumor cells. The use of tissue microarray (TMA) in combination with immunohistochemistry (IHC) may be a valuable approach to validate the predictive and diagnostic utility of various biomarkers in non-cancerous tissues like placental tissue.

Transport phenomena with reactions in drug delivery: Towards a quantitative description

AbstractA distributed system called a Krogh cylinder is used here to quantify the transport of a solute from the capillary into the extravascular tissue. The capillary network is broken down into cylindrical cells, each containing a capillary and an appropriate amount of extravascular tissue. The flow in the cylinder model has two‐dimensional velocities, which are in the axial and radial directions. All parameters of the system, together with the geometric ones, have been included in the model. For a given bioavailability, the uptakes of reactive and nonreactive solutes have been obtained. Very large or massive molecules have been considered. The diffusion in the tissue is found to be very low. Most of the drug uptake happens through convection, which is slowed down in the presence of a reaction. Further, this convection is entirely due to the flow out into the lymphatic system. For the case where a reaction takes place, local equilibrium is assumed, which both cuts down the computation times and provides good results in the case of reactive solutes. The full results of a distributed system have been obtained for the first time, and the mechanics of how the area‐under‐the‐curve can be used to calculate the actual solute uptake have been determined.

Red blood cell dynamics in extravascular biological tissues modelled as canonical disordered porous media.

The dynamics of blood flow in the smallest vessels and passages of the human body, where the cellular character of blood becomes prominent, plays a dominant role in the transport and exchange of solutes. Recent studies have revealed that the microhaemodynamics of a vascular network is underpinned by its interconnected structure, and certain structural alterations such as capillary dilation and blockage can substantially change blood flow patterns. However, for extravascular media with disordered microstructure (e.g. the porous intervillous space in the placenta), it remains unclear how the medium's structure affects the haemodynamics. Here, we simulate cellular blood flow in simple models of canonical porous media representative of extravascular biological tissue, with corroborative microfluidic experiments performed for validation purposes. For the media considered here, we observe three main effects: first, the relative apparent viscosity of blood increases with the structural disorder of the medium; second, the presence of red blood cells (RBCs) dynamically alters the flow distribution in the medium; third, symmetry breaking introduced by moderate structural disorder can promote more homogeneous distribution of RBCs. Our findings contribute to a better understanding of the cell-scale haemodynamics that mediates the relationship linking the function of certain biological tissues to their microstructure.

Preoperative and intraoperative risk factors for post‐anaesthetic pulmonary oedema in horses

SummaryBackgroundPulmonary oedema is an uncommon but life‐threatening condition in horses. It is defined as an abnormal accumulation of liquid and solute in the extravascular tissues and spaces of the lungs, which causes oxygen diffusion impairment and a reduction in lung compliance. Diagnosis is determined by typical clinical signs, including frothy, haemorrhagic nasal discharge and respiratory distress, and can be supported by thoracic imaging and arterial blood gas analysis. In horses, post‐anaesthetic pulmonary oedema (PAPOE) is poorly reported, and there is a lack of information regarding risk factors and prognosis.ObjectivesThe objective of the study was to determine risk factors and short‐term outcome for PAPOE during the recovery period in horses.Study designA retrospective case‐control study including 17 cases and 53 controls.MethodsMedical records of clinical cases and controls over a period of four and a half years were reviewed. Short‐term outcomes and risk factors for PAPOE were investigated using conditional multivariable logistic regression analysis.ResultsAscarid impaction prevalence in PAPOE cases was significantly higher than controls (p = 0.03). In a univariable analysis, the following variables were significantly associated with PAPOE (p ≤ 0.05): gastric reflux on admission, haemorrhage during surgery, intravenous crystalloid treatment prior to admission, fresh frozen plasma and hydroxyethyl starch administration during anaesthesia. On multivariable analysis, gastric reflux on admission (OR = 7.9, 95% CI 1.1, 57.2) and fresh frozen plasma treatment during anaesthesia (OR = 9.6, 95% CI 1.2, 79.1) were found as risk factors for PAPOE. There was no significant difference in outcome between cases and controls.Main limitationsThe low number of cases, the lack of gross and histological post‐mortem examinations of the horses that did not survive and the clinical definition of PAPOE.ConclusionsGastric reflux and fresh frozen plasma administration during general anaesthesia are associated with PAPOE.